Ligilactobacillus Salivarius improve body growth and anti-oxidation capacity of broiler chickens via regulation of the microbiota-gut-brain axis

Certain strains of probiotic bacteria can secret functional substances namely digestive enzymes and functional peptides to regulate physiological conditions such as digestion and anti-oxidation, which are often incorporated in industrial broiler chick production. However, few studies have detailed the action mechanisms and effects of these bacteria on regulating growth and anti-oxidation levels in broiler chickens. Ligilactobacillus salivarius is a strain of probiotic bacteria used as dietary supplement. In the present study, Ligilactobacillus salivarius was evaluated for its secreted digestive enzymes in vitro. To detailed evaluate the action mechanisms and effects of gastrointestinal tract (GIT) microbiota on alleviating anti-oxidation levels of broiler chickens through the gut-brain axis. Ligilactobacillus salivarius was cultured and supplemented in the food of broilers to evaluate the probiotic effect on growth and anti-oxidation by modulation of gut microbial composition and its functional metabolites using metagenomic and metabolomic assays. Biochemical results showed that Ligilactobacillus salivarius secreted digestive enzymes: protease, lipase, and amylase. Broiler chickens with Ligilactobacillus salivarius supplemented for 42 days, showed increased body weights, a reduced oxidative status, decreased malondialdehyde levels, and improved activities rates of total superoxide dismutase, glutathione peroxidase IIand IV improved. The microbial composition of caecum was more abundant than those broiler without probiotics supplementation, owing 400 of total number (489) of bacterial operational taxonomic units (OTU). The genera of Lactobacillus, Megamonas, Ruminoccoccaceae, Ruminococcus, Alistipes and Helicobacter shared the dominant proportion of Candidatus _Arthromitus compared with the control chickens. These functional bacteria genera assisted in the transportation and digestion of amino acids, carbohydrates, and ions, synthesis of cellular membranes, and anti-oxidation. Uncultured_organism_g_ Anaerosporobacter, Lactobacillus salivarius, uncultured_bacterium_g_ Ruminococcaceae_UCG-014, uncultured_bacterium_g_ Peptococcus were strongly and positively correlated with body growth performance and anti-oxidation. A metabonomic assay suggested that the secreted of gamma-aminobutyric acid and monobactam was metabolized according to the Kyoto Encyclopedia of Genes and Genomes analysis. In conclusion, Ligilactobacillus salivarius optimized microbial composition of the caecum and secreted functional peptides through gut-brain axis to improve the body growth and antioxidation of broiler chicken.


Introduction
In broiler chicken industrial production, several unfavourable environmental conditions, including high concentrations of harmful gases, stocking density, heat, cold, and transportation, represent adverse factors for body health; these factors induct a decrease in growth and immunity, a disturbance of the gastrointestinal bacterial composition, and increased secretion of cortisol [1][2][3].Probiotics are defined as live microorganisms that [4], when administered in adequate amounts, provide a myriad of benefits to body health, such as modulating immunity, improving the gut's bacteria composition, and reducing oxidative levels through the secretion of functional substances [5,6].Probiotics are considered a useful additive substitute for antibiotics in industrial broiler chicken production [7,8].Some strains of probiotic bacteria can secrete digestive enzymes to help nutritional absorption including γ-aminobutyric acid [9], endorphin [10], and monobactam [11], which regulate brain functions through the gut-brain axis.The hormone secreted such as cortisol is affected by the composition of the microbiota to modulate anti-oxidation, as regulated by the hypothalamic-pituitary-adrenal axis [12].
Ligilactobacillus salivarius (L.salivarius) is a probiotic strain widely used in the food and diet of humans and animals.We have previously demonstrated dietary supplementation with 10 6 colony-forming units of probiotics per gram of diet (CFU/g) [13].Dietary supplementation with L. salivarius can decrease the level of stress-correlated oxidation in animal production [6,13].In addition, the gastrointestinal bacterial composition can be optimized via supplementation with L. salivarius [14].However, its beneficial effects in broiler chickens remain unclear.The functional substances related to the antioxidant defence of L. salivarius are unknown.Moreover, the connection between bacterial composition in the gastrointestinal tract (GIT) and the secretion of hormones to regulate oxidation in broiler chickens has rarely been studied.
Metabolomic analyses have been widely used to identify unknown microbial metabolites [15].Functional substances have often been identified, annotated, and quantified.Metagenomic assays are often employed to evaluate the intestinal bacterial composition and its relationship with body growth and other factors [16].In this study, the secretion of digestive enzymes by L. salivarius was examined using a plate assay.Broiler chickens were fed a L. salivarius supplementation to increase their body weight.Biochemical analyses were performed to measure hormone secretion and oxidative status.To further detail the action pathway of L. salivarius, the optimized composition of the microbiota in the caecum was verified using a metagenomic assay, and metabolomics were used to identify functional substances.The purpose of our study was to elucidate the potential action mechanism of the microbiota of the GIT with L. salivarius supplementation through the gut-brain axis, as this mechanism may provide a new avenue for exploring the effects and potential connections of probiotic bacteria in practical use.

Materials and methods
Bacterial strain and digestive enzyme assay L. salivarius was isolated by our research group at the School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry; it was then stored at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with strain number CGMCC17718.The bacteria were cultured and colonised in de Man, Rogosa, and Sharpe medium (MRS) after inoculation at 1% incubation and 37℃ for 16 h of cultivation.After cultivation, the fermented medium was filtered at 0.45 μm to remove the bacteria.The fermentation liquid of L. salivarius was collected to measure the secretion of digestive enzymes.Whole milk powder (2 g) was added to the MRS medium to prepare the plated medium.An Oxford diffusion assay was employed using the Oxford diffusion method [14].The biochemical colorimetric method was employed to quantify the capacity of the digestive enzyme; the live bacteria after fermentation were calculated and diluted to 10 9 colony forming units per millilitre (CFU/mL), then filtered; the residual liquid was investigated for protease, lipase, and amylase activities secreted by L. salivarius.Detection kits were purchased from Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China.

Chickens, diet, and study design
To evaluate the supplemented efficiency of L. salivarius, 360 one-day-old Cobb broilers (average body weight 40.13 g purchased from Jiangsu Lihua Animal Husbandry Co., Ltd.) were randomly allocated to four groups with five replicates of 18 each.These were the control (CON), L. salivarius (LSA), vitamin C (VIT), and flavomycinsupplemented (FLA) groups.All animal management and euthanized in experiments were approved by the Institutional Animal Care and Use Committee of China and the Institution of Animal Science and Welfare of Jiangsu Province (no.IASWJSP202111739).The study was conducted in accordance to relevant guidelines and regulations.All efforts were obeyed the rules of animal welfare and were to minimize animal sufferings.All the authors confirm that the study is reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org).
Chickens in the control group were fed a basal diet, whereas those in the three treatment groups were fed a basal diet supplemented with L. salivarius, vitamin C [17] and flavomycin.The experimental diets were fed during two periods, designated as starter (days 0-21) and finisher (days .The basal diet composition, which did not include any probiotics or antibiotics, as well as the nutrient analysis results are shown in Table 1.All nutrients met or exceeded the nutrient requirements of the National Research Council (NRC, 2012) [18].For chickens in the vitamin C group, 1.12 g of vitamin C (analytically pure) was diluted in 100 mL distilled water and blended with 3 kg of the basal diet.Thereafter, the mixed basal diet was added to a blender containing 97 kg of the basal diet.The blender was used for 10 min to ensure uniform mixing.For the L. salivarius group, 50 mL of L. salivarius fermentation liquid was measured separately and blended with 3 kg of the diet, followed by a mass diet of 97 kg.After preparation, 5 g of the diet was harvested to detect live bacteria using the plate method.The sample was treated with a series of ten dilutions; a 100 µL dilution was spread on the plate of the MRS solid medium.The number of live L. salivarius reached 2.5 × 10 6 colony forming units per gram (CFU/g) of diet.The diet for the flavomycin group was prepared using 2 g of premixed food containing 20% flavomycin blended with 100 kg of the basal diet to reach a concentration of 4 mg/kg.All chickens were allowed ad libitum access to water and food throughout the experimental period.

Sample collection
The chicks in each replicate of each treatment group were weighed on day 42.Their daily dietary consumption was accurately recorded.After 42 d, three chickens with an average body.
weight in each replicate were selected (n = 5 × 3) and subjected to fasting for 12 h.Samples were harvested under general halothane anaesthesia (purchased from Pfizer Incorporated, USA) followed the dose of 0.65 mg per kilogram bodily wight.A 5 mL volume of blood was obtained from the wing vein and homogenised with heparin sodium to harvest plasma after centrifugation at 3000 rpm.The caecum tissues were removed under aseptic conditions, stored in sterile plastic tubes on ice, and immediately transported to our laboratory for deoxyribonucleic acid extraction.A segment of 3 cm ileum samples from the distal end of the ileum to the ileocecal orifice was collected and stored in 4℃, then the intestine was sectioned longitudinally and washed with cold physiological saline solution (4℃) to remove chyme.A 0.2 g sample of mucous membrane was shaved and ground to prepare a mucosal tissue homogenate.

Hormonal levels and antioxidant status
Plasma samples were used to measure the hormone levels.Plasma cortisol [19], endotoxin [20], and antioxidative indices, namely malondialdehyde (MDA), superoxide dismutase (SOD), and activities of type II glutathione peroxidase (GPX) [21], were detected using enzymelinked immunosorbent assay (ELISA).The activity rate of type IV GPX [22] in the caecal mucous membrane was measured using ELISA kits.These kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Table 1 Nutrient analysis of the basic diet for broiler chicks
Note: The premix provides, a Vitamins and trace elements per kg diet: Vitamin A (retinyl acetate) 9, 875 IU, Vitamin D 3 (cholecalciferol) 3, 000 IU, Vitamin E (DL-ɑ-tocopheryl acetate) 20 IU, menadione 3.25 mg, Vitamin B 12 (cyanocobalamin) 0.025 mg, thiamin 1.5 mg, riboflavin 5.0 mg, biotin 0.032 mg, folacin 1.25 mg, niacin 12 mg, pantothenic acid 12 mg, and pyridoxine 3.75 mg, manganese 100 mg, zinc 80 mg, iron 80 mg, copper 8 mg, iodine 0.15 mg, and selenium 0. Diversity metrics were calculated using the core diversity plugin in QIIME2 [23].Feature-level alpha diversity indices and operational taxonomic units (OTUs) were used to estimate microbial diversity within an individual sample.A co-occurrence analysis between the anti-oxidative status, stress level and bacterial species in caecal chyme was performed by calculating Spearman's rank correlations and producing network plots.Additionally, the potential Kyoto Encyclopedia of Genes and Genomes (KEGG) [24] orthologue functional profiles of the microbial communities were predicted using PICRUSt.

Metabolomic assays of functional peptides
A fermented liquid without L. salivarius cells was prepared.Then, 100 µL of a fermented medium with L. salivarius were added to 800 µL of extract liquid composed of methanol and acetonitrile (volume ratio of 1:1) containing 0.02 mg/mL 2-chlorophenylalanine as an interior label.These substances were subsequently vortex mixed for 30 s and subjected to low temperature ultrasonic extraction (5℃, 40 kHz).The samples were refrigerated at -20℃ for 30 min, then centrifuged at 13,000 rpm/min for 30 min.Subsequently, the supernatant was reconstituted with a 120 µL water resolution containing 50% acetonitrile and transferred into a vial for ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS) analysis [25].Twenty microlitres of each sample to be tested were mixed with a quality control (QC) sample for error correction.Certain metabolising substances, such as monobactam, were identified by searching freely accessible KEGG, which is a database that integrates genomic, chemical, and systemic function information [26].
To qualify the concentrations of gamma-aminobutyric acid (ɤ-GABA) and monobactam in the fermented liquid of L. salivarius, the standard of ɤ-GABA (Alladdin Company, USA) and monobactam (Pfizer, USA) were purchased and diluted into 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, and 1000ng/mL concentrations to establish the standard curve.The assays were performed following the UPLC-MS procedure [27].

Statistical analyses
Body weight and DNA sequencing data (Shannon index) were subjected to one-way ANOVA using the GLM procedure in SPSS, with significance reported at P < 0.05.The means were further separated using Duncan's multiple range test [28].A P-value of less than 0.05 was considered statistically significant.

Secretion of digestive enzymes
An obvious zone of plaque was observed on the milk MRS medium plate, as indicated by the Oxford diffusion results shown in Fig. 1.An obvious dissolved zone was observed in the outer space of the Oxford cap.The diameters of the zones reached 18.29 mm.The capacities of protease, lipase, and amylase secreted by L. salivarius were 178.89U/mL, 698.29U/mL, and 532.43U/mL, respectively at 10 9 CFU/mL.

Impact on growth performance
The growth performance is shown in Fig. 2.After supplementation with L. salivarius for 42 d, the broiler chickens displayed a higher average body weight than those of the control by 268.93 g (Fig. 2a) (P < 0.05).However, this was not significantly different from the average weight of chickens who received the flavomycin supplementation (P > 0.05).Body weights in the vitamin C supplanted groups were not significantly different from that of the control group (P > 0.05).The average daily gain was the same (Fig. 2b).Broiler chickens inoculated with L. salivarius and flavomycin were higher than those in the control (P < 0.05).There were no differences in the average daily feed intake among the treatments (Fig. 2c) (P > 0.05).The ratio of feed:gain in the vitamin C and control groups was nearly 0.18 units higher than those in chicks with the L. salivarius and flavomycin supplements (Fig. 2d) (P < 0.05).

Hormonal levels and antioxidant status
Cortisol levels in the plasma of chicks are listed in Table 2, with L. salivarius supplementation significantly decreasing in all treatments (P < 0.01).The result in the vitamin C group was also lower than those in the control and flavomycin groups (P < 0.01).The levels of endotoxins were also reduced in the L. salivarius-supplemented group (P < 0.01).
Plasma antioxidant indices were also measured.The MDA content in chickens supplemented with L. salivarius significantly decreased, and the activity rates of SOD and type II GPX were higher than those of the vitamin C and flavomycin groups (P < 0.05), which were enhanced compared with the control (P < 0.05).The activity rates of type IV GPX in the L. salivarius-and vitamin C-supplemented groups also improved compared to those of the control and flavomycin groups (P < 0.05).

Optimized bacterial composition in cecum
The caecal chyme microbial 16 S rRNA metagenome was sequenced, and the results are shown in Fig. 3.The bacterial composition in the L. salivarius group was the highest, and the number of OTUs reached 400, covering 81.79% of the total of 489 (Fig. 3a).The number of patients in the control group was 317, which was the lowest of all groups (P < 0.001).The OTU diversity was in the order L. salivarius > vitamin C > flavomycin > control.The composition of bacteria in the Fig. 2 Effects of different treatments on growth performance of broiler chicken.The final body weight, average daily gain (ADG), average daily feed intake (ADFI), and ratio of feed: gain (F: G).Chicks were treated with control (CON), vitamin C (VIT), Ligilactobacillussalivarius (LSA), and flavomycin (FLA) after 42 days.Data was statistically processed as one-way ANOVA using the GLM procedure of SPSS, with significance reported at P < 0.05.Bars represent mean ±S.E.* is significantly different from those without (P<0.05).Figure 2a.Effects of different treatments on chicken body weight.Figure 2b.Effects of different treatments on chicken average daily gain (ADG).Figure 2c.Effects of different treatments on chicken average daily feed intake (ADFI).Figure 2d.Effects of different treatments on chicken ratio of feed: gain (F: G) caecum showed significant differences between L. salivarius and flavomycin supplementation through alpha diversity (Shannon index) at both the phylum and genus levels (Fig. 3b  and d).At the phylum level, the dominant bacteria in the control group were Firmicutes, and there were a few other bacterial phyla observed.The chicks in the supplemented groups had higher abundances of Epsilonbacteraeota (P < 0.001), Actinobactria (P < 0.05), Tenericutes (P < 0.01), and Lentisphaerae (P < 0.01), compared to the controls.The bacterial abundance in the L. salivarius group was the highest.No significant differences were observed between the vitamin C and flavomycin groups.At the species level, the abundance of the three supplementary groups improved, and the Shannon indices shown in Fig. 3c suggested an increased diversity (P < 0.01).The richness of uncultured_bacterium_ gFaecalibacterium, uncultured _bacterium_g_Megamonas, gut_metagenome_g_Desulfovibrio, Ruminococcaceae, and unclassified_f_Lachnospiraceae were higher in the three supplementary groups than in the control groups.The abundances of unclassified_f_Lachnospiraceae and uncultured _bacterium_g_Megamonas were the highest (P < 0.01).gutmetagenome, uncultured_bacterium_g_Megamonas Ruminococcaceae in the L. salivarius groups (P < 0.01).Uncultured bacterium_g_Romboutsia and Desulfovibrio contained in flavomycin group were higher than those in the other groups (P < 0.01).The abundance of Gallibacterium_anatis was the highest in the vitamin C group.

Functions of the optimised bacterial composition
The functions of the optimised bacterial genes were predicted and classified using the KEGG pathway, as shown in Fig. 4. Certain function-related genes, such as energy production and conversion, nucleotide transport and metabolism, and cell membrane biogenesis were increased in L. salivarius and vitamin C-supplemented groups compared with chicks in the control group.The abundances of two kinds of genes, transcription and signal transduction mechanisms, were the highest in the L. salivarius group compared to the other three groups.

Functional metabolites
Functional metabolites secreted by Ligilactobacillus salivarius were detected using UPLC-MS; ɤ-GABA and monobactam were measured.The effective concentrations of ɤ-GABA and monobactam reached 382.19 and 456.93 ug/L, respectively.The biosynthetic pathway was characterised using the KEGG classification, as shown in Fig. 6.The pathway originate from the biosynthesis of phenylalanine, tyrosine, and tryptophan through cascade-catalysed reactions.These reactions are divided into two pathways: the metabolism of L-arginine, and continuous syntheses that were merged into the metabolisms of glycine, serine, and threonine.One pathway of alanine, aspartate, and glutamate biosynthesis also influenced the synthesis of 3α-Hydroxy-3-aminoacyl-momobactamic acid, the precursor of monobactam.The product was completed in a further step.

Discussion
Numerous studies have suggested that probiotic bacteria can promote livestock animals production.However, some studies have reported contrasting results [29,30].

Table 2 Effects of different treatment on body anti-oxidation and hormonal levels
Mean value was significantly different from that of the control group by one-way ANOVA followed by Tukey's multiple comparison tests: the different superscript capital letters in the same color of column mean significant difference at 0.01 levels (P < 0.01), lowercase letters means.significantdifference at 0.05 levels (P < 0.05) Fig. 3 The overall profile of microbiota composition in cecal digesta of chicks.Figure 3a The Venn map on the OTU of microbiota in digesta of cecum.Figure 3b Shannon index on bacterial differences in phylum level.Figure 3c One-way ANOVA on bacterial composition in level.Figure 3d Shannon index on bacterial differences in species level.Figure 3e One-way ANOVA analysis on bacterial composition in species level.* in same column means P < 0.05, ** means P< 0.01, *** means P < 0.001 Therefore, the benefits of candidate probiotic bacteria as a supplement for chicks should be further evaluated.L. salivarius is a probiotic bacterium that is often used in the diets of humans and animals [31,32].L. salivarius was isolated and supplemented in the diet to detect effects on performance and the action pathway to evaluate its use in industrial broiler chicken production.

Groups
The digestive enzymes metabolised by L. salivarius were first measured.These measurements suggested that three types of enzymes-protease, lipase, and amylase-could be secreted.In broiler chickens, insufficient feed digestion and nutrition absorption cause a waste of feed some nutrients in the diet cannot be digested or excreted in faeces and are, therefore, wasted [33].When supplemented with a dose of 10 6 CFU/g L. salivarius in the digestive enzymes can be by gut bacteria and used to promote digestion in broiler chicks, thereby improving digestion efficiency and nutrition utilization [6,14].
The digestive enzymes produced represent one of several advantages of L. salivarius [31].The optimal bacterial composition in the GIT was determined.Results of alpha diversity on bacterial community showed that the OTUs from chicks supplemented with L. salivarius were richest, both in phylum and species levels.Increased abundance and types of bacteria at both the phylum and species levels were detected in caecal chyme with the L. salivarius supplementation.The bacterial proportions were evenly distributed.The proportion of Firmicutes to Bacteroidetes was reduced to 3.5:1, which was healthier than the control and appropriate for intestinal digestion and metabolism [32].The diversity of bacterial differences at the species level is also shown.Unclassified_f_Lachnospiraceae and uncultured _bacterium_g_Megamonas were the most abundant in the L. salivarius group.Faecalibacterium, Megamonas, gut_metagenome_g_Desulfovibrio, Ruminococcaceae, and unclassified_f_Lachnospiraceae increased in richness.All these improved bacterial species demonstrate improved digestion and absorption [34,35], leading to enhanced growth performance and immunity [36,37].
Fig. 4 The functional analysis on bacterial composition.Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway function classification.KEGG is a database resource that integrates genomic, chemical, and systemic functional information.STAMP software was applied to detect the differentially abundant KEGG pathways among groups with false discovery rate correction.Certain functions related genes: energy production and conversion, nucleotide transport and metabolism, cell membrane bio-genesis were increased The correlation analysis of bacterial species suggested that the increased abundances of Ruminococ-caceae_UCG-014, unculture_bacterium_g_Peptococcus, gut_metagenome_g_ Desulfovibrio, unclassified_g_But yricicoccus, and unclassified_f_Lachnospiraceae led to improved body growth and lower anti-oxidation levels.The improved bacterial composition at both the phylum and species levels was crucial for the probiotic role of L. salivarius [31].
The prediction for genetic functions of the composition of the microbiota indicated that with L. salivarius supplementation, energy production and conversion, nucleotide transport and metabolism, cell membrane biogenesis, transcription, and signal transduction mechanisms improved.Dietary supplementation with L. salivarius can regulate body stress and minimize oxidative damage to cellular membranes.The bacteria were previously recognised by toll like receptors (TLRs) of the microbiological pattern recognition receptor, activated nuclear transcription factor kappa B (NF-κB), through the classic signalling pathway TLR-NF-κB [38].The gutbrain axis was regulated by the microbial composition of the GIT.Oral supplementation of L. salivarius regulated brain functions.
Body hormone secretion was regulated by brain.In suitable physiological condition, levels of cortisol and adrenaline synthesized in adrenal gland will be upregulated to overcome the stress [39,40], accompanied with high levels of bodily anti oxidative status.In industrial breeding and production, unfavourable environmental conditions often cause adverse factors to chicks [41].Supplemented with L. salivarius can reduce the secretion of cortisol and oxidative levels.To unveil the related metabolites produced by the bacteria, the metabolomic assays were employed, and the metabolic profiles of L. salivarius in relation to stress mitigation were also studied.The secretions of ɤ-GABA and monobactam appear to represent another mitigation factor.ɤ-GABA is a neurotransmitter in the central nervous system [42], and performs the biological roles of improving sleep quality, inhibiting excitement, and lowering blood pressure; its action mechanisms have been widely clarified, leading to its widespread usage [43].Monobactam is another functional peptide secreted by L. salivarius that facilitates antihypertension, analgesia, and anti-convulsion benefits [44].It has been used as an antibiotic drug in recent clinical studies [45].The production of monobactam via the chemical pathway is expensive and intensive, and can be synthesised through microbiological paves [46].The results showed that the metabolisms of phenylalanine, tyrosine, tryptophan, L-arginine, glycine, serine, and threonine all influenced monobactam synthesis.
These two peptides are secreted into the gut during bacteria colonisation and are then assimilate into the blood and transported to the brain.This influences hormone secretion through the gut-brain axis [47].Hence, the stress-related hormones cortisol and endotoxin decreased in chicks supplemented with L. salivarius.The results of the antioxidative indices SOD and types II, and IV of GPX indicated an improved anti-oxidation status [48].The mitigated stress induced by higher body antioxidation is attributed to a lower oxidative status [49].The concentration of MDA, an oxidation productin plasma also declined [50].

Conclusion
Ligilactobacillus salivarius secret digestive enzymes in its metabolism.A dietary supplementation with 2.5 × 10 6 CFU/g L. salivarius can help broiler chicken digest and nutrition absorption, establish optimized caecal bacterial composition, also its syntheses of ɤ-GABA and monobactam absorbed into brain to modulate the secretion of stress-hormone cortisol though Fig. 6 The profile of Monobactam on Ligilactobacillus salivarius.The biosynthesis pathway was annotated depending on KEGG.The graph of metabolic pathway was found through enriched of KEGG metabolic pathway after collecting the metabolites through UPLC-MS detection.Monobactam produced by L. salivarius and their pathway were manifested comprehensively.The pathway was originated from the biosynthesis of phenylalanine, tyrosine, and tryptophan through the cascade catalyzed reactions, which divided into two pathways, one is the metabolism of L-Arginine, the other way was continuously biosynthesized and merged into metabolism of glycine, serine, and threonine.One pathway of alanine, aspartate, and glutamate biosynthesis is also involved to synthesize 3α-Hydroxy-3-aminoacyl-momobactamic acid the precursor of monobactam, and finish the synthesis in further step the gut-brain axis, reduce the oxidative levels, inducing to improved body growth (Fig. 7).Our study unveiled the effects and elucidated the potential action mechanism of L. salivarius supplementation through the gutbrain axis, which provided a new avenue for evaluating and exploring the potential action of probiotics in practical use.

Fig. 1
Fig. 1 The Oxford diffusion assay on the digestive enzymes secreted by Ligilactobacillus salivarius.The obvious dissolved zone can be observed around the Oxford cap.The diameter of the zones was 18.29 mm